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Notes Two New Lactones from Cephalotaxus fortunei var. alpnia Jiang Du, Ming-Hua Chiu,* and Rui-Lin Nie Kunming Institute of Botany, Academia Sinica, Kunming 650204, People’s Republic of China Received January 22, 1999
Two new lactones, fortunolides A (1) and B (2), together with three known compounds, hainanolidol,32 acetylcephalotaxine, and wilsonine, were isolated from the needles and stem of Cephalotaxus fortunei. Their structures were elucidated on the basis of MS and NMR data. The genus Cephalotaxus is composed of 10 species, eight of which are distributed in China.1 Investigations of the chemical constituents of extracts of Cephalotaxus sp. resulted in the isolation of a number of alkaloids. Several of these, such as harringtonine, isoharringtonine, and homoharringtonine,2 are of interest due to their antitumor activity. Sun and co-workers3 reported two nonalkaloid compounds, named hainanolide and hainanolidol, with a new ring skeleton from Cephalotaxus hainanensis. Hainanolide has cytotoxic activities against L-615, S180, W256, P-388, and L-1210 cells. In this paper, we report the chemical constituents of Cephalotaxus fortunei Hook. f. var. alpnia Li. Five compounds, fortunolide A (1), fortunolide B (2), hainanolidol (3)3 acetylcephalotaxine, and wilsonine,4,5 were isolated from stems and needles of this plant with the aid of aluminum oxide column chromatography. Fortunolides A (1) and B (2) and hainanolidol belong to the hainanolide3 skeletal type. All structures were identified by NMR and MS methods, and 1 and 2 were two new compounds. Only four compoundsshainanolide, hainanolidol, 1, and 2sare known from higher plants that possess the hainanolide skeleton.
The IR data of 1 (C19H20O4) indicated tropone and lactone moieties, whose presence was supported by UV absorptions at 317.5 and 252 nm, respectively. Comparing the 1H and 13C NMR spectrum of 1 (Table 1) and hainanolide,3 we found that these two compounds have the same carbon skeleton. The 1H NMR spectra showed the presence of two methyls, connecting with tertiary and troponic carbons, respectively. The two protons of the tropone appeared at δ 6.67, 6.68 (J ) 2.0 Hz) and showed long-range couplings, indicating that they are in the same plane with a Wconformation. The 13C NMR data also supported the existence of tropone and lactone. Fragment CH (CH3)CH2-CH-CH-CH2 was elucidated on the basis of the 1H1H and 1H-13C COSY spectra. The IR spectrum showed a * To whom correspondence should be addressed. Tel.: 086-871-5150660, ext. 3255. Fax: 086-871-5150227. E-mail:
[email protected].
10.1021/np9900270 CCC: $18.00
Table 1.
13C
and 1H NMR Data (δ) for Compounds 1 and 2 1
no.
13C
1 2 3 4 5 6 7 8 9 10 11 12 13R 13β 14R 14β 15 16 17 18 19
186.0 140.8 147.0 146.2 145.1 143.8 138.8 28.4 17.9 49.7 86.8 45.6 35.7
a
28.8 77.2 173.5 30.0 19.3 24.4
2 1H
13C
186.3 141.5 146.0 145.6 145.4 143.8 6.67 (d, J ) 2.0 Hz) 139.5 2.52, 3.08 (m, R, βH) 32.3 1.76, 2.65 (m, R, βH) 22.8 47.7 58.4 2.83 (m) 82.0 2.43 (dd, J ) 19.0, 3.0 Hz) 89.7 3.28 (dd, J ) 19.0, 9.5 Hz) 1.46 (m) 84.8 1.84 (m) 4.71 (t, J ) 4.4 Hz) 81.6 173.5 1.41 (q, J ) 6.8 Hz) 41.7 0.90 (d, J ) 6.9 Hz) 14.7 2.15 (s) 23.6 6.68 (d, J ) 2.0 Hz)
1H
7.10 (d, J ) 2.0 Hz)
6.99 (d, J ) 2.0 Hz) 2.75, 2.83 (m, R, βH) 1.20, 2.40 (m, R, βH) 3.59 (s) 5.39 (s) 4.12 (d, J ) 6.0 Hz) 5.48 (d, J ) 6.0 Hz) 1.77 (q, J ) 7.6 Hz) 0.79 (d, J ) 7.6 Hz) 2.23 (s)
Compound 1 in C5 D5N; 2 in CDCl3.
quaternary hydroxyl group (3500-3300, 1150 cm-1). Based on the proposed skeleton of the compound, it was concluded that this hydroxyl group could only be at C-11 or C-12. The 1H-3C COSY spectrum showed that H-12 was split to a deformed triplet. So one hydroxyl group, linked with C-11 rather than C-12, was confirmed. The 1H-1H COSY spectrum of 1 showed that H-12 coupled with H-13β and that H-15 significantly correlated with H-12, H-17, and H-14β, suggesting that H-12, H-15, and H-17 were in β-positions. The R- and β-protons linked with C-13 were doublet doublets with different coupling constants (J ) 19.0, 3.0 Hz and J ) 19.0, 9.5 Hz). This phenomenon can be explained as follows: the R- and β-protons at C-13 couple with each other and split into two doublets (J ) 19.0 Hz), which further couple with H-12, respectively. The coupling constant of 9.5 Hz indicated an axial coupling between the proton at C-12 and the β-proton at C-13, and J ) 3.0 Hz indicated an axial-equatorial coupling between the H-12 and H-13R. The conformation of 1 was proposed based on the COLOC spectrum. Both C-11 and C-16 coupled with the H-15, from which we can propose that two six-membered rings, composed of C-10, C-11, C-12, C-14, C-15, and C-17 and C-10, C-14, C-15, C-16, and C-17, are both in the boatlike conformation. On
© 1999 American Chemical Society and American Society of Pharmacognosy Published on Web 10/27/1999
Notes
the basis of the above analyses, the structure of fortunolide A is determined as 1. All of the1H and 13C NMR signals were unambiguously assigned on the basis of 2D NMR experiments. The spectral data of 2 (C19H18O5) also indicated the presence of lactone and tropone moieties.1H and 13C NMR signals suggested that 2 has a structure similar to hainanolide. The only difference between them was that 2 had one additional oxygen and an extra degree of unsaturation. After comparing the NMR spectra of the two compounds (2 and hainanolide), it was concluded that the extra hydroxyl group in 2 was at either C-11 or C-12. In the 1H NMR spectrum of 2, the H-13 signal is a singlet significantly downfield, so the hydroxyl group must be linked with C-12. Otherwise, the H-13 signal would split into a doublet by H-12. H-14 coupled with the H-15, and they both were doublets (J ) 6.0 Hz). However, the coupling constant between H-17 and H-14 was too small to be seen in the 1H NMR spectrum measured at 400 MHz. These observations indicated that both H-14 and H-15 are axial (J ) 6-14 Hz) and that H-17 is equatorial. Thus, the structure of fortunolide B was determined to be 2. Hainanolidol was isolated in 1979.3 By means of 1H-1H and 13C-1H COSY spectra, we have confirmed the structure of hainanolidol and have assigned all of the 1H and 13C NMR signals. Experimental Section General Experimental Procedures. Melting points were measured with a WC-1 melting point apparatus and were uncorrected. IR spectra were recorded with a Perkin-Elmer 577 spectrometer. UV spectra were obtained with a Shimadzu UV-210A spectrophotometer. MS were measured with a VGAUTOSPEC mass spectrometer. Optical rotations were obtained on a JASCO DEP-370 polarimeter. 1H, 13C, and 2D NMR spectra were recorded using a Bruker AM-400 instrument. Plant Material. Stems and needles of C. fortunei Hook.f. var. alpina Li were collected in Weixi County of Yunnan Province, China. The plant was identified by Assistant Professor Lianfang Li, Forestry Institute of Sciences of Yunnan, China. Extraction and Isolation The air-dried stems and needles (4.5 kg) were extracted with cold 95% EtOH. The solution was
Journal of Natural Products, 1999, Vol. 62, No. 12 1665
concentrated, and an equal volume of 2% aqueous HCl was added. The insoluble part was separated from the acidic solution by filtration and further extracted twice with 2% aqueous HCl. The combined acidic solutions were neutralized using solid Na2CO3, then extracted with CHCl3 to afford 14 g of crude extract. The crude extract was subjected to repeated aluminum oxide column chromatography to yield compounds 1 (36 mg), 2 (21 mg), hainanolidol (16 mg), acetylcephalotaxine (57 mg), and wilsonine (30 mg), with yields of 8.0 × 10-4, 4.7 × 10-4, 3.6 × 10-4, 1.3 × 10-3, 6.7 × 10 - 4 % from the airdried plant material (4.5 kg), respectively. Fortunolide A (1): obtained as pale yellow powder [(CH3)2CO]; mp 263-265 °C; [R]22.5 D +136.11°(c 0.709, CHCl3); UV (CHCl3) λmax (log ) 252 (4.23), 317.5 (3.90) nm; IR (dry film)νmax 3600-3300 (w), 3100-3200, 2940, 2900, 1740, 1615, 1580, 1525, 1440, 1070, 1010, and 910 cm-1; 1H and 13C NMR, EIMS (70 eV) m/z 312 [M+ ], (74), 298 (17), 284 (99), 266 (83), 251 (45), 238 (47), 223 (72), 185 (60), 167 (71), 160 (100), 115 (65), 108 (54), and 55 (88); HREIMS m/z 312. 1362 (calcd for C19H20O4, 312.1376). Fortunolide B (2): obtained as yellow needles [(CH3)2CO]; mp 248-250 °C; [R]18.6 D +181.82°(c 0.506, C5H5N), UV (CH3OH) λmax (log ) 248.5 (4.26), 320 (3.82) nm; IR (dry film)νmax 3300-3000 (w), 2960, 1750, 1615, 1590, 1530, 1365, 1170, 1145, 1070, 1025, 990, and 875 cm-1; 1H and 13C NMR, EIMS (70 eV) m/z 326 [M+], (37), 298 [M+ - H2O], (67), 218 (32), 130 (100), 103 (21), 77 (27); HREIMS m/z 326.1154 (calcd for C19H18O5, 326.1123). Hainanolidol (3): obtained as yellow powder [(CH3)2CO]; mp 226.5-227.5 °C; UV (CHCl3) λmax (log ) 251.5 (4.26), 322 (4.02) nm; IR (dry film)νmax 3500-3100 (w), 2943, 2918, 1745, 1610, 1515, 1426, 1375, 1293, 1050, and 890 cm-1; 1H and 13C NMR; EIMS (70 eV) m/z 312 [M+], (75), 284 [M+ - H2O], (77), 221 (62), 207 (97), 196 (90), 181 (54), 165 (64), 153 (67), 143 (100), 128 (62), 115 (53), and 95 (75). References and Notes (1) Wu, C. Y. Index florae Yunnanensis; The People’s Publishing House of Yunnan: Kunming, 1984; Vol. 1, p 23. (2) Mikolajczak, K. L.; Powell, R. G., Smith, C. R. Tetrahedron 1972, 28, 1995-2001. (3) Sun, N. J.; Xue, Z.; Liang, X. T., Huan, L. Acta Pharm. Sinica 1979, 1439-1443. (4) Powell, R.G.; Mikolajczak, K. L.; Weisleder, D.; Smith, C. R. Phytochemistry 1972, 11, 3317-3320. (5) Du, J.; Qiu, M.; Nie, R. Nat. Prod. Res. Dev. 1997, 9, 19-24.
NP9900270
